PROJECT SUMMARY Chromosomal rearrangements involving the KMT2A/Mixed Lineage Leukemia gene (MLL-r) drive the development and sustained growth of an aggressive form of Acute Myeloid Leukemia (AML). Small molecule inhibitors of the DOTL1 methyltransferase and MENIN effectively suppress MLL-fusion driven oncogenic gene expression, however, early clinical studies indicate that resistance will limit clinical utility and highlights an urgent need for effective combination therapies to improve patient outcomes. Here a functional genomic screening was undertaken to elucidate resistance mechanisms and synthetic lethal dependencies during treatment with inhibitors of either MENIN or DOT1L and identified IKZF1/IKAROS as a putative therapeutic target to improve the efficacy of these drugs; implicating an important role for IKAROS in maintaining the MLL-r oncogenic transcriptional program. The immunomodulatory (IMiD) drugs, including Lenalidomide, drive degradation of IKAROS, providing an immediate route to implementing a novel combination therapy with MENIN and DOT1L inhibitors. Preliminary data suggests that the combination therapy of MENIN inhibition and an IMiD compound results in synergistic killing of MLL-r AML cell lines. This proposal aims to (1) characterize the function of IKAROS within the transcriptional program of MLL-r AML and (2) evaluate IKAROS as a novel therapeutic target in MLL-r AML and characterize the synergistic effect of the combination MENIN inhibitor/IMiD treatment as it relates to pathways of IKAROS degradation. The use of a combination of genetic, interactomic, proteomic, mouse models and compound-induced targeted protein degradation approaches will enable an understanding of the oncogenic contribution of IKAROS and a mechanistic understanding of the effectiveness of combination treatments. This work will capitalize on expertise in the biology of MLL-r AML and collaborative work on IMiD compound development and optimization to establish new effective combination therapies for MLL-r AML. This work will build upon and improve the burgeoning field of therapeutic uses of targeted protein degraders. The worked described here will continue to develop my scientific skill set and is in line with my career goals of dissecting mechanisms of protein regulation in leukemia using cutting edge technologies from multiple disciplines. To that end, a comprehensive training program has been developed to maximize the potential to become an independent investigator with a unique approach toward protein science research in the context of disease.